Regulation of Gene Expression within the Locus for Enterocyte Effacement

Enteropathogenic and enterohemorrhagic Escherichia coli (EHEC and EPEC respectively), in common with many other pathogenic Gram-negative bacteria of veterinary or human importance, utilise type-III secretion to subvert eukaryotic signalling pathways by injecting bacterial effector proteins into the host cell cytoplasm. Within these pathovars, a well-characterised type-III secretion system (TTSS) encoded by the locus for enterocyte effacement (LEE) is responsible for the development of the attaching-effacing lesion and for other effects on enterocyte function. Regulation of the LEE is complex and is governed by many different regulatory influences. Recently, work in the Pallen laboratory has shown that two regulators, EtrA and EivF, encoded within a second type-III secretion cluster (the ETT2 gene cluster) exert a profound effect on gene transcription in the LEE in EHEC (but not in EPEC). However, we have yet to identify the mechanism. This regulatory cross-talk might arise through direct effects on LEE promoters, but is equally likely to be the result of indirect effects mediated though other regulators.<P>
Thus, a principal goal of this proposal is the identification of the direct targets of EtrA and EivF using the novel cutting-edge technologies of chromatin immunoprecipitation and microarrays. Chromatin immunoprecipitation (ChIP) is a method for determining the in vivo binding sites of a given regulatory protein and thus provides an invaluable tool in unravelling regulatory networks and sorting direct from indirect effects, particularly when allied with microarray technology. ChIP studies will be followed by probing investigations into the details of relevant protein-DNA and protein-protein interaction. <P>In addition, we will adopt similar approaches to a selection of other known or suspected regulatory influences on the LEE. Candidates include GrlA, GrlR and the cytoplasmic FHA domain of EscD, all in encoded within the LEE, and the alarmone, ppGpp (we have preliminary evidence that secretion of LEE-encoded proteins is up-regulated in a RelA mutant of EHEC). Aside from chromosomal genes, plasmid- encoded genes represent another potential source of regulatory influence on the LEE. Recent creation of a plasmid-cured derivative of the EHEC O157:H7 Sakai strain in Birmingham, twinned with local availability of microarrays, provides us with a unique opportunity to examine the effects of plasmid loss on gene transcription within the LEE.